In recent years, the microarray technology has emerged as an important technique with wide applications in different areas of biomedical analysis thanks to its versatility and miniaturization capability. In order to accomplish a large number of genomic or proteomic tests in parallel, a microarray substrate is functionalized with thousands of probes bound to a solid surface in pico or femto moles (10−12, 10−15 amounts. As a consequence, the physical and chemical characteristics of the interfacial layer between the biomolecules and the inorganic substrate are key factors for the success of the technique. In fact, proper biomolecule deposition and immobilization are crucial to increase the accuracy of data analysis as spot morphology has a great impact on the signal level: non homogeneous spots, such as doughnut-shape spots or coffee ring effect may cause errors in the acquisition and measurement of signals, thus affecting the reliability of the assay [1]. Furthermore, advances on micro fabrication techniques have led to the development of miniaturized and fully integrated solid phase analytical devices that imply a scaling down of the entire analytical system and process while maintaining high sensitivity. The miniaturization of the system requires excellent control of the surface properties in order to maximize probe immobilization in reduced areas, avoiding spot merging and cross contamination.
Several strategies have been adopted to control spot morphology and merging. In particular, considering the drying rate of a diluted droplet placed on a surface, hybrid hydrophobic/hydrophilic substrates have been introduced: Moran-Mirabal and collaborators have solved the problem of coffee ring effect by fabricating a polymer liftoff surface which combines a hydrophobic polymer film of PARYLENE, (chemically vapour deposited poly(pxylylene) polymer) with photolithographically patterned hydrophilic areas of a Self Assembled Monolayer (SAM), where the DNA is deposited. The part of the drop in contact with the hydrophobic area undergoes, through evaporation, an inward capillary flow which reduces the coffee stain effect [2]. Similarly, Lee and collaborators introduced a hybrid substrate constituted of two different types of SAMs one hydrophobic and one hydrophilic, obtained, similarly, through photolithography [3]. Even though these approaches solve the issue of irregular spots and cross-contamination, facilitating the assay analysis, they are very complicated to perform and involve the use of SAMs which show several drawbacks such as thermal instability, limited range of functional groups displayed on the surface, long reaction times to obtain monolayers [4]. Furthermore, the two-dimensional arrangement of SAM restricts the maximal surface density of the functional moieties and provide limited accessibility of functionalities [5]. Three-dimensional surfaces, instead, provide a homogenous surface presenting high concentration of reactive groups, resulting in an increased binding capacity of targets compared to monodimensional films [5]. Ultimately, they act as linkers distributing the bound probe also in the axial position, thus causing a faster reaction with the target involved in biomolecular recognition. Recently, a 3D-hydrogel was presented as a method to immobilize DNA, in order to increase probe density [6]. The hydrogel is photopolymerized onto a particular support, called PolyShrink, a thermosensitive material, which offers the possibility of reducing the array dimensions by heating the surface. As a consequence, also the height of the spot increases (up to 6 μm), promoting hybridization, but the immobilization procedure is quite complicated as it consists in the deposition of a mixture of a pre-synthesized polymer together with the oligonucleotides, which are then photopolymerized; the entire array is then cured at 160° C. to shrink the surface, a temperature which is not compatible with several biological systems.
In 2004, Pirri and collaborators [7] proposed a polymer coating realized by a combination of physisorption and chemisorption that promotes the attachment of biomolecules by exposing functionalities such as active esters reactive towards the nucleophile groups of proteins, peptides or amino-modified DNA. In particular, this copolymer, poly(DMA-NAS-MAPS), is constituted of three monomers: N, N-dimethylacrylamide (DMA) that binds to the surface by weak non-covalent interactions such as hydrogen bonding, Van der Waals or hydrophobic forces, N-acryloyloxysuccinimmide (NAS) a chemically reactive monomer that covalently binds DNA and proteins to the surface, and a pending silane hydrolyzable monomer, 3-(trimethoxysilyl) propyl methacrylate (MAPS), which promotes the condensation of the polymer with surface silanols. The coating is obtained by simply immersing the support (glass [7], silicon oxide [8] [9], nitrocellulose [10], gold [11], plastics [12]) in a diluted aqueous solution of the polymer. Beside the simplicity of its use, another peculiar characteristics of this coating is its hydrophilicity, which results in high resistance to nonspecific binding and low background signal [12].
Unfortunately, the hydrophilicity of a surface has important consequences on the process of deposition of liquid droplets. When the distance between spots must be kept low to increase spot density, it is important to control how the liquid spreads out over the surface as well as the size of the spot. This latter parameter is related to the hydrophobicity of the surface as measured by the contact angle. A low contact angle (<45°) indicates a hydrophilic surface with good wetting properties on which water readily spreads and sticks. A high contact angle (>90°) denotes a hydrophobic surface where water does not interact forming droplets that do not stick to the surface but are easily displaced. A desirable coating must prevent excessive spreading of the droplets to concentrate the probe in small areas. However, a firm attachment to the underlying surface is also needed so that the droplets remain in position on the surface and dry-out to produce a spot with a reproducible size and uniform intensity. In some circumstances, the poly(DMA-NAS-MAPS) coating provides a surface which is too hydrophilic to allow the creation of an array of small sizes with reduced spot-to-spot distance: reduced spotting areas are, in fact, typical in miniaturized biosensors, microfluidic devices, and LoC (Lab on a Chip).
Additional background is provided in priority U.S. provisional application 62/114,486 filed Feb. 10, 2015, which is incorporated herein by reference, including reference to the following technical documents: EP Patent 1 567 569 B1; U.S. Pat. No. 8,809,071; Cretich M. et al., Functionalization of Poly(dimethylsiloxane) by Chemisorption of Copolymers: DNA Microarrays for Pathogen Detection, Sens. and Actuat. B-Chem., 2008, 132, 258-264; and Brown and Botstein, Nature Genetics Supplement Vol 21, 1999, pp. 33-37.
Hence, despite advances in the art, a need yet exists for better polymer structures which can be used, for example, in thin, nanoscale layers for biomolecular binding and molecular surface assays, including high density microarrays.